The secretion of neurotransmitters, neuropeptides and peptide hormones occurs by Ca2+dependent vesicle exocytosis. The machinery that mediates vesicle fusion with the plasma membrane is well-characterized and consists of complexes of the vesicle SNARE VAMP-2/synaptobrevin with the plasma membrane SNAREs syntaxin-1 and SNAP-25. SNARE complexes are acted upon by Ca2+bound synaptotagmin to drive membrane fusion. While these final steps in Ca2+triggered vesicle fusion have been extensively studied, there remain important gaps in understanding key events that precede fusion. These events are termed priming and they confer competence to docked vesicles for Ca2+triggered fusion. Priming is thought to involve the progressive assembly of trans SNARE complexes but the precise pathway utilized and the factors that regulate it have not been characterized. Genetic and biochemical studies indicate that members of the CAPS/Munc13 family of proteins operate in priming. Our proposed research will obtain a molecular description of the mechanism of CAPS function in priming dense-core vesicle (DCV) exocytosis. This work is based on major advances during the previous project period, which discovered that CAPS promotes trans SNARE complex formation in vitro and interacts with each of the 3 SNARE proteins. Moreover, CAPS undergoes tetramer formation, which suggests a mechanism for catalyzing SNARE complex assembly. CAPS is present on the plasma membrane and on DCVs in a central location for controlling DCV exocytosis. We propose biochemical, cell biological and biophysical studies on the molecular events that mediate CAPS function in priming. Our specific aims will be to: (1) determine whether the priming activity of CAPS is mediated through its interactions with VAMP-2, syntaxin-1 and SNAP-25;(2) determine whether CAPS promotes trans SNARE complex assembly in neuroendocrine cells;and (3) determine whether CAPS oligomerization and DCV binding play critical roles for CAPS function in priming. Highlights of the work include imaging of CAPS and SNAREs at sites of exocytosis, reconstituting priming by CAPS on artificial membranes, and structural studies of CAPS tetramers and its SNARE-binding domain. Completion of this work will provide a molecular description of priming and fill an important gap in our understanding of the pathway for regulated vesicle exocytosis. PUBLIC HEALTH RELEVANCE: Neurotransmitters, peptide hormones, and inflammatory mediators are secreted from neural, endocrine and mast cells by the regulated fusion of vesicles with the plasma membrane. We will study key proteins that are needed for this process. Because a variety of diseases involve excessive or inadequate secretion, this work will contribute basic knowledge about these conditions and set the stage for developing therapies.